A photo-caged platinum(II) complex that increases cytotoxicity upon light activation

Article abstract of DOI:10.1002/ejic.201000098

A novel platinum(II) photocaged complex called [Pt(cage)] has been prepared and characterized by X-ray crystallography. The complex contains a photolabile nitrophenyl group incorporated into the backbone of a tetradentate ligand that contains two pyridyl and two amide nitrogen donor sites. The intact complex is unreactive toward ligand-exchange reactions until activation with UV light cleaves the ligand backbone, releasing a Pt^II complex that more readily exchanges its ligands, as verified by reaction with a methioninc-containing peptide. [Pt(cage)] is non-toxic to MCF-7 cells in the dark, whereas brief UV exposure induces cell death of human breast cancer MCF-7 cells at a level approaching that of cisplatin. By using light to alter the coordination chemistry around the metal center, [Pt(cage)] represents a new strategy for potentially delivering metal-based drugs in a site and time specific manner.

Full text of DOI:10.1002/ejic.201000098

SHORT COMMUNICATION
DOI: 10.1002/ejic.201000098
A Photo-Caged Platinum(II) Complex That Increases Cytotoxicity upon Light
Activation
Katie L. Ciesienski,[a] Lynne M. Hyman,[a] Daniel T. Yang,[a] Kathryn L. Haas,[a]
Marina G. Dickens,[a] Robert J. Holbrook,[a] and Katherine J. Franz*[a]
Keywords: Platinum / Cisplatin / Caged compounds / Phototherapy / Photoactivation / Prodrugs / Cytotoxicity
A novel platinum(II) photocaged complex called [Pt(cage)]
has been prepared and characterized by X-ray crystallogra-
phy. The complex contains a photolabile nitrophenyl group
incorporated into the backbone of a tetradentate ligand that
contains two pyridyl and two amide nitrogen donor sites. The
intact complex is unreactive toward ligand-exchange reac-
tions until activation with UV light cleaves the ligand back-
bone, releasing a PtII complex that more readily exchanges
its ligands, as verified by reaction with a methionine-contain-
ing peptide. [Pt(cage)] is non-toxic to MCF-7 cells in the
dark, whereas brief UV exposure induces cell death of hu-
man breast cancer MCF-7 cells at a level approaching that
of cisplatin. By using light to alter the coordination chemistry
around the metal center, [Pt(cage)] represents a new strategy
for potentially delivering metal-based drugs in a site and
time specific manner.
drawback of the PtIV complexes reported to date is their
sluggish photoactivation, which can take upwards of an
hour of UV irradiation.
Introduction
Since the initial discovery of cisplatin’s cytotoxicity by
Rosenberg in the 1960s, platinum-based drugs, which now
include carboplatin and oxaliplatin as well as cisplatin, have
become a cornerstone of modern anticancer chemotherapy
regimens.[1] The clinical effectiveness of these agents, unfor-
tunately, is restricted by dose-limiting toxicity and intrinsic
or acquired resistance of some tumors. Significant efforts
to understand the cellular response and tumor resistance
mechanisms of platinum drugs[2,3] have inspired the devel-
opment of new compounds to overcome these limitations.
Strategies include altering the coordinating ligands on plati-
num, designing drug delivery systems that distribute plati-
num compounds selectively to tumor cells, and developing
prodrugs that release cytotoxic agents following an acti-
vation step.[4,5] In this area, substitution-inert PtIV agents
that are reduced intracellularly to active PtII compounds
are attractive for diminishing off-target cytotoxicity and re-
sistance.[5] More recently, Sadler and co-workers introduced
photoactive PtIV prodrugs in which the reduction occurs
only upon illumination with UV light.[6–8]
As an alternative to photoactive PtIV compounds, we
present here a novel photo-caged PtII complex in which a
photoactive nitrophenyl group is incorporated into the li-
gand backbone. The tetradentate chelating ligand sup-
presses ligand-exchange reactions to provide a nontoxic PtII
complex, [Pt(cage)]. Activation with light induces bond
cleavage of the ligand, as shown in Scheme 1, which con-
verts neutral [Pt(cage)] into a charged and exchange labile
PtII complex 3. Both properties may be beneficial for cellu-
lar retention of the photolyzed compound. Importantly,
photoactivation occurs within minutes and induces signifi-
cant photo-dependent cytoxicity.
Photoactive nitrophenyl groups have been used in count-
less examples to block (i.e. “cage”) the biological activity of
a variety of molecules such that exposure to UV light turns
on a biological function.[11,12] This strategy is also being
explored to develop complexes that allow photoinitiated
drug release for applications in photochemotherapy.[10,13–16]
The concept of light-activated caged metal ions was first
Photoactive compounds that can deliver cytotoxic drugs
intracellularly only at the irradiated site can potentially in-
crease the specificity of a drug and thereby minimize its
toxicity to surrounding healthy tissue.[9,10] Such a strategy
is envisioned for surface-associated diseases or for internal
introduced for Ca2+ [17]
panded to biologically important d-block metal ions like
Cu2+ and Zn2+ [18–20]
Metal complexes themselves have also
,
and has only recently been ex-
.
been exploited as caging groups, for example ruthenium
polypyridines that release bioactive compounds by light-in-
duced ligand dissociation.[21,22] In these contexts, the termi-
nology “cage” implies photocage, where light is used to ef-
fect a molecular change that alters a biological response. It
does not imply a geometric configuration and is in fact dis-
tinct from the classical inorganic definition of cage, which
refers to a polycyclic compound having the shape of a cage,
tissue accessible via endoscopic fiber optic technology.[9]
A
[a] Department of Chemistry, Duke University,
P. O. Box 90346, Durham, North Carolina 27708, USA
Fax: +1-919-660-1606
E-mail: katherine.franz@duke.edu
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201000098.
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Photo-Caged Platinum(II) Complex
Scheme 1.
or an inclusion compound. The report here demonstrates UV/Vis spectrophotometry. As shown in Figure 2, spectral
that a photoactive ligand can be used to unleash the cytoto- changes are apparent within seconds of illumination and
xicity of a metal-based agent.
exhibit an increase at 320 nm that is characteristic of a ni-
troso photoproduct. These data suggest that the photoreac-
tion and in turn cleavage of the ligand backbone is com-
plete in approximately 2 min. Analysis of the reaction mix-
ture by liquid chromatography-mass spectrometry (LC-MS)
revealed 3 as the major product (see Supporting Infor-
mation), implying that ligand cleavage occurs at two sites.
The products are slightly different from those observed for
the apo-ligand or its CuII complex, where the ligand is
cleaved only at one position.[18] We hypothesize that upon
excitation of the nitrophenyl group, initial bond cleavage
occurs to release the picolinamide fragment shown coordi-
nated to Pt in 3 and a nitroso photoproduct that sub-
sequently undergoes a Norrish type II photoreaction to lib-
erate the imine fragment bound to Pt in compound 3 along
with nitroso-containing by-products (see Supporting Infor-
mation). The quantum efficiency for the photolysis of
[Pt(cage)] was determined to be 0.75 (Supporting Infor-
mation). The ligand itself has a quantum efficiency of 0.73,
which indicates that coordination by PtII does not decrease
photolysis efficiency, as previously observed for [Cu-
(OH2)(cage)].[18]
Results and Discussion
[Pt(cage)] was prepared in one step by combining equi-
molar quantities of H2cage[18] and Na2PtCl4 in basic ethan-
ol. Recrystallization from acetone/H2O permitted analysis
by X-ray crystallography that confirmed that two deproton-
ated amide nitrogens and two pyridyl nitrogens coordinate
the PtII center in square-planar geometry, as shown in Fig-
ure 1. In comparison, the copper complex of this ligand,
[Cu(OH2)(cage)], has distorted trigonal bipyramidal geome-
try that can be attributed to a steric interaction between the
α hydrogens on the pyridyl rings.[18] The increased radius of
PtII compared to CuII alleviates this steric clash to accom-
modate the square planar arrangement preferred by this d8
metal ion. The average Pt–amide bond length of 1.987 Å
and Pt–pyridyl distance of 2.036 Å are similar to those of
[Cu(OH2)(cage)], 1.943 Å and 2.034 Å respectively.
Figure 2. UV/Vis spectra of 80 µ [Pt(cage)] in phosphate buffer at
pH 7.4 photolyzed in 10 s intervals for a total of 180 s (initial spec-
trum at time zero is dashed line); inset: absorbance at 320 nm vs.
irradiation time shows that the compound is completely photolyzed
within 2 min.
Figure 1. ORTEP plot of [Pt(cage)] showing 50% thermal ellip-
soids. Selected bond lengths and angles: Pt–N1, 2.036(6); Pt–N2,
1.979(6); Pt–N3, 1.994(7); Pt–N4, 2.036(6) Å; N2–Pt–N3, 93.7(3);
N2–Pt–N1, 79.8(3); N3–Pt–N1, 173.4(3); N2–Pt–N4, 173.7(3); N3–
Pt–N4, 80.2(3); N1–Pt–N4, 106.4(3)°.
The photolysis experiments described above indicate that
two bidentate photoproducts remain coordinated to PtII
To investigate the photoreactivity of [Pt(cage)], solutions following illumination. In order to determine whether such
of the complex dissolved in pH 7.4 phosphate buffer were a product could induce cell death, we treated human breast
irradiated in a Rayonet photoreactor and monitored by carcinoma MCF-7 cells with [Pt(cage)] and monitored cyto-
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K. J. Franz et al.
SHORT COMMUNICATION
toxicity of irradiated vs. non-irradiated samples. For com- synergy between the metal component and ligand compo-
parison, cells were also treated with cisplatin. As shown in nent that could be further exploited to improve light-acti-
Figure 3, control cells exposed to 2 min of UV light remain vated cell killing.
viable over the course of 96 h and display no increase in
cell death compared to cells kept in the dark. As expected,
cisplatin induces cytotoxicity in a dose-dependent manner
with concentrations ranging from 50–200 µ. Exposure of
cisplatin-treated cells to UV light only subtly increases sen-
sitivity of the cells to cisplatin cytotoxicity. On the other
hand, cells treated with up to 200 µ [Pt(cage)] and left in
the dark remain viable over the 96 h timecourse, whereas
those that are also exposed to 2 min of UV irradiation show
a significant increase in cell death. These results suggest
that intact [Pt(cage)] itself is non-toxic, but activation with
UV-light releases photoproducts that induce cell death in a
light-dependent fashion.
Figure 4. Results of LDH cytotoxicity assay performed on MCF-
7 cells treated with 5–300 µ H2cage and either left in the dark or
exposed to UV light for 2 min prior to 48 h incubation.
The binding of platinum drugs to DNA is believed to
be the primary biological interaction responsible for their
anticancer properties.[2] In an attempt to visualize such in-
teractions for [Pt(cage)] or its photoproducts, we used aga-
rose gel electrophoresis. Intact [Pt(cage)] at concentrations
up to 300 µ had no effect on the migration of either circu-
lar or linear plasmid DNA through the gel, suggesting that
the compound does not react with DNA to form platinum
adducts, at least under the conditions tested (see Supporting
Information). This result was expected, given the stability
of the tetradentate chelator designed to minimize ligand ex-
change reactions. However, photolyzed samples of
[Pt(cage)] also failed to cause a shift in DNA migration.
The lack of evidence for light-dependent DNA platination
Figure 3. Results of cytotoxicity assay performed on MCF-7, hu-
requires further investigation in order to understand the
man breast carcinoma cell line. Cells were treated with 5–200 µ
biological activity of 3 that induces the cytotoxicity ob-
cisplatin or [Pt(cage)] and either left in the dark or exposed to UV
light for 2 min (+ UV). Cell death was assessed by the LDH release served in Figure 3. Notably, other tetraamine PtII com-
plexes have shown cytoxicity in cancer cell lines.[23]
assay after 48, 72, or 96 h, as indicated. Control cells (ctrl) received
no drug treatment but were exposed to UV light.
PtII interactions with sulfur-containing biomolecules are
believed to play various roles in the uptake, excretion, resis-
Control experiments using H2cage show that the ligand tance and toxicity of platinum drugs.[24] Current Pt-based
itself is cytotoxic, even in the dark. As shown in Figure 4, drugs are thought to enter cells by a combination of passive
a 200-µ dose of H2cage causes nearly 60% cell death diffusion and facilitated uptake by transporters that include
within 48 h, even in the absence of UV irradiation. Cytoto- the sulfur-rich copper transport protein Ctr1. We, and
xicity further increases when the cells also receive 2 min of others, have shown that the methionine-rich extracellular
UV exposure, even at the earliest timepoint monitored in regions of Ctr1 are capable of coordinating Pt drugs and,
these experiments (48 h). In contrast, cells treated with especially in the case of cisplatin, inducing complete loss of
200 µ [Pt(cage)] and irradiation remain mostly viable at the carrier ligands.[25–27] Because such interactions are likely
48 h, showing less than 15% cell death. The combined re- to diminish the cytotoxic potential of Pt drugs, we were
sults shown in Figure 3 and Figure 4 indicate that coordina- curious to compare the difference in reactivity of [Pt(cage)]
tion to PtII actually mitigates an inherent cytotoxicity of pre- and post-photolysis with a model Ctr1 peptide. To
the ligand. Furthermore, the timing discrepancy in light- show that photolysis of [Pt(cage)] causes a change in its
activated cell killing implies different mechanisms of toxic- reactivity we monitored the ability of the complex to react
ity for [Pt(cage)] and H2cage, which photolytically decom- with the peptide AcMMMMPMTFK that we have pre-
pose into different organic fragments. The observations that viously shown reacts with cisplatin, carboplatin, and oxali-
the carrier ligand’s toxicity is increased by light-activation platin.[25] As shown by the LC/MS data in Figure 5, intact
but can be masked by metal binding suggests a possible [Pt(cage)] shows no interaction with AcMMMMPMTFK
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Photo-Caged Platinum(II) Complex
even after 24 h of incubation at 37 °C. On the other hand, a neutral to charged complex may be another beneficial
photolyzed samples show a decrease in signal intensity for property imparted by the photoactivation of [Pt(cage)] in
both 3 {compared to photolyzed [Pt(cage)] samples without addition to the tetradentate-to-bidentate conversion.
peptide} and the peptide (see Supporting Information, Fig-
ure S4), suggesting that a reaction has occurred. Further-
Conclusions
more, new peaks emerge in the UV chromatogram that indi-
cate a complex mixture of products. While most of these
species were not able to be identified, one provided a strong
ion peak in the mass spectrum at m/z 1382.5, which is con-
sistent with formation of [Pt(AcMMMMPMTFK)]+. This
result further confirms that the intact [Pt(cage)] is inert to
ligand subsitution, whereas the photoproduct can react
with biomolecules and shed the bidentate ligands.
In conclusion, we have presented an inert PtII compound
that upon irradiation with UV light uncages a biologically
active PtII complex by cleavage of the ligand backbone.
[Pt(cage)] was shown to be non-toxic to human breast carci-
noma MCF-7 cells in the dark, however upon irradiation
its cytotoxicity increased by 65%. [Pt(cage)] will be a valu-
able tool for delivering Pt intracellularly in a site and time
specific manner and represents an alternative strategy for
activating metal-based drugs with light.
Experimental Section
CCDC-756957 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Supporting Information (see also the footnote on the first page of
this article): Experimental procedures.
Acknowledgments
This work was supported in part by the Camille and Henry Dreyfus
Foundation, the Sloan Foundation, the National Science Founda-
tion (CAREER 0449699) and Duke University. D. Yang acknowl-
edges a fellowship from the Beckman Scholars Program. Thanks
to Ms. Amanda Hoertz for assistance with DNA samples.
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